Fisher John P. e.a. (ed.) Tissue Engineering
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14.3.3 Physical Delivery Methods
A highly efficient way of transferring naked DNA into a select cell or group of cells is via physical
transfection methods. Microinjection by a skilled operator offers nearly perfect delivery efficiency on a
cell-by-cell basis. This technique can be used to transfer oligonucleotides to cell cytoplasms or nuclei [58],
or entire nuclei into enucleated eggs, as is the case with cloning [59,60]. However, the use of microinjection
to transfect hundreds or thousands of somatic cells in vivo would be an infeasible venture, both in terms of
getting to and visualizing the cells of interest, and the amount of time and effort required for the procedure.
There exist alternative physical delivery methods that can be used to deliver genetic material directly into
a larger number of cells, albeit in a slightly less precise fashion. These methods include electroporation
and the gene gun.
Since 1982, electroporation has been a promising approach for both in vitro and in vivo gene
delivery [61]. Electroporation causes disruption of the plasma membrane and formation of membrane-
associated DNA aggregates, which enter the cytoplasm through transient pores [62]. These complexes
have been shown to enter the cytoplasm 30 min after administration of current, and proceed to peri-
nuclear locations by 24 h postadministration [62]. Electroporation has shown promising results in the
administration of complexes to skeletal muscles, indicating a possible role for the technique in future
in vivo muscular applications [63]. Electroporation can be combined with chemical transfection meth-
ods, including dendrimers, to increase the overall efficiency of transfection [64]. A similar approach,
termed nucleofection, utilizes an electrical current in conjunction with specific solutions to deliver DNA
not only into the cell but also into the cell nucleus [65].
The gene gun provides a ballistic means of transporting genetic material into cells. A typical application
of the gene gun would entail attaching DNA to gold particles that are on the order of 1 µm in diameter.
The coated gold is then loaded onto a cartridge or onto a disc and propelled down the barrel of the gun
via pressurized helium at a force on the order of 200 to 300 psi. The end of the gun barrel is too small
for the disk to exit, so its motion is halted abruptly before exit. The coated gold colloids detach from
the disc and continue their path, passing through or becoming embedded within cells. When passing
through cell membranes, cytoskeletons, and other structures, some of the DNA can become dislodged
and remain within the cells for processing. This method provides good transfection of tissue surface layers
and offers an advantage over microinjection in that many cells in a specified area are quickly transfected.
However, cell damage and depth of gold penetration are two limiting factors of this method. The gene
gun has been used successfully in vivo to transfect murine tumors with cytokines, successfully restricting
cell growth [66]. Direct gene gun transfer of gold-adhered DNA particles has also been used in successful
transfections of beating hearts with genes encoding the green fluorescent protein [67].
14.4 Intracellular Pathways
The mechanisms governing the transport of nonviral gene delivery complexes into the cytoplasm and
on into the nucleus vary between different vectors. Endocytosis is the primary means for cellular import
of nonviral gene carriers, which makes complex modification through the addition of specific ligands
beneficial. Endocytosed complexes enter the cytoplasm in endosomes, which mature from early to late
endosomes before meeting up with lysosomes to form endolysosomes. As this vesicle maturation occurs,
membrane bound H
+
/ATPases create an acidic environment within the compartments. It is this acidic
environment which allows lysosomal enzymes (acid hydrolases) to remain active and potentially degrade
the endocytosed material. The endolysosome therefore presents a major obstacle to nonvirally mediated
gene therapy, as evidenced by increases in transfection efficiency in the presence of the lysosomotrophic
agent chloroquine [68–70]. Research into lysosomal disruption has produced a peptide that can degrade
the membrane of the late endosome, based on its cholesterol content. This method has reportedly increased
the transfection efficiency of parenchymal liver by 30-fold [71].
PEI has been hypothesized to serve as a proton acceptor within endolysosomes, serving to buffer the
pH of the endolysosome while additional H
+
is pumped in. According to this hypothesis, Cl
−
ions will
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Gene Therapy 14-7
leak into the vesicles to alleviate the electric gradient, thereby creating an osmotic gradient which will be
offset by an influx of water molecules into the endolysosomes. The result will be swelling and bursting
of the endolysosomes [45]. PEI has been shown to enter cells via endocytosis, and the endosomes have
been shown to swell within a period of under 6 h [72]. However, there exists a question of the degree
of lysosomal involvement during PEI-mediated transfection. PEI/DNA-containing endosomes have been
imaged while passing through lysosomal beds without lysosomal interaction [73]. In addition, PEI has
been reported to utilize microtubules of the cytoskeleton as a direct pathway to the nuclear envelope,
thereby eluding lysosomes [74]. It is perhaps the large excess of positive charges in PEI/DNA complexes
that help direct the complexes in a seemingly atypical fashion during cellular processing. More research is
required to elucidate the complete cellular mechanism of PEI-mediated transfection.
If the delivered genetic material is DNA, and the desired outcome of the transfection is expression of
the delivered gene, then the next major hurdle to successful transfection is getting the delivered DNA
into the cell nucleus for transcription. Many studies have monitored the effect of cell cycle stage upon
transfection efficiency. Some viral vectors show strong transduction during all phases of the cell cycle
because of their strong nuclear-importation machinery [75]. However, polyplex and lipoplex carriers
show optimal transfection if they are administered during the G1 or S phases of the cell cycle [75]. It is
known that the nuclear envelope is dismantled during late mitosis, hence perhaps these findings can be
explained by the length of time required for transfection complexes to proceed from endocytosis to the
outer nuclear membrane being roughly equal to the amount of time needed for the cell to progress from
G1 or S phase to late mitosis. Other work indicates the barrier to transfection presented by the nuclear
envelope through results that reveal very low transfection efficiencies in nondividing cells when cationic
lipid-mediated vectors were used [76].
Aside from entering the nucleus by virtue of location during mitotic nuclear membrane disruption,
genes and gene delivery complexes can be delivered to the nucleus via import. Possible mechanisms
for nuclear import include interactions between the complexes and other proteins bound for nuclear
import and interactions between the complexes and nuclear import receptors themselves. Proteins in the
cytoplasm that are bound for nuclear import typically have a conserved peptide sequence that is recognized
by the cell as a nuclear localization signal (NLS). Such signals can be manufactured in the laboratory for
inclusion as an integral part of the gene delivery complexes [77,78]. Many factors govern the success of
NLS-mediated nuclear import: the type of NLS, the manner in which the NLS is incorporated, DNA form,
and the proper definition of the complexes [79].
14.5 Cell and Tissue Targeting
Each of the physical delivery methods listed in Section 14.3.3 is an excellent way to deliver genes to a
specific region. Certainly, microinjection is a very straightforward way to introduce genes into a single cell
of interest. However, physical delivery methods can be technically difficult, insubstantial in the number of
cells that are transfected, or impossible for delivery to remote areas of the body. To provide feasible
alternatives when such challenges exist, molecular targeting methods have been developed, yielding
encouraging results.
The use of cell-specific ligands or receptors to target extracellular attributes is a good method for
gene delivery to specific cell types or classes. Neuronal cells have been targeted for transduction via
a neuron-specific viral envelope in conjunction with specific glycoproteins, leaving nonneuronal cells
unaffected [80]. Other examples of ligands that have been used include LOX-1 to potentially target
endothelial cells [81], the human hepatitis B virus preS1 peptide to target hepatocytes [82], plus transferrin
[83], folate [84], anti-CD34 [85], and galactose [86], among others. The list of potential and realized target
ligands is very large, which suggests the utility of this targeting technique.
Using cell-specific transcription regulation sequences (binding elements such as promoters or enhan-
cers) is another way to target cells without modification of the gene delivery vehicle, which allows for cell
targeting with known delivery and release kinetics without having to repeat the same work for modified
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vectors. This type of targeting, known as promoter or expression-targeting, works on the principle that
many cell types could take up the delivered genes, but only the cells of interest will transcribe them because
the transcriptional machinery that binds to the utilized promoters is present only in the target cells. This
type of targeting has been used to target megakaryocytes via the promoter for integrin αIIβ (for a possible
treatment for platelet disorders) [87]. Targeting liver cells has been achieved by using the promoter for
liver-type pyruvate kinase and SV40 enhancer [88]. Expression-targeting has also been used to target an
entire class of cells (Cox-2 overexpressing cells), which could be an important technique in the treatment
of carcinomas or inflammation [89]. The use of two specific binding elements, a promoter/enhancer
combination, has been employed to deliver the suicide gene for thymidine kinase to prostate tumors [90].
Expression-targeted gene delivery could prove to be a valuable tool in clinical gene therapy, alone or in
conjunction with ligand-targeted systems.
14.6 Applications
14.6.1 In Vitro
In 1977, a chemically created somatostatin gene was fused to an Escherichia coli β-galactosidase gene
and introduced into a bacterium resulting in expression of the gene [91]. The alteration of both cells and
cellular functions has expanded greatly since then to produce many new experimental applications. In vitro
modeling is valuable in learning more about living systems without the involvement of animals, while
potentially leading to in vivo applications. For example, liposomal delivery of the gene for adenomatous
polyposis coli (APC), delivered to produce a potential anticancer agent, has produced excellent results in
diminishing a human duodenal cancer in the presence of bile in vitro [92].
The proliferation of cells in vitro not only serves as a model for in vivo studies but also serves as a
valuable component of most ex vivo applications. However, it is often the case that cell behavior differs
between in vitro and in vivo environments. Hematopoietic stem cells show this characteristic, displaying
substantial proliferation in vivo but dividing at a more conservative rate in vitro. Building on the retroviral
overexpression of the homeobox B4 gene, a 40-fold increase in in vitro growth has been observed in
mouse bone marrow cells [93]. However, the difference between cell behavior inside and outside the
living organism along with the involvement of a wider array of cell types and cellular proteins continue to
be significant barriers to scientific research, and are reasons why in vitro investigations are not enough to
produce treatments ready for the clinic. In vitro research is still important because it allows greater control
over experimental conditions for molecular studies and reduces the need for research animals.
In vitro gene therapy also has applications in the creation of regulation vehicles for the complexes
needed in vivo. The delivery of l-dopa and dopamine to the nervous system by genetically altered cells
can be regulated by embedding the cells in hydrogels, created and currently being tested in vitro, and
implanting these systems into the affected area [94]. Another regulation device has been created from
freeze-dried PEI/DNA complexes along with sucrose and PLGA to create a porous medium filled with
encapsulated transfection complexes [95]. In vitro, the use of this PLGA sponge has caused expression of
a reporter gene in adherent fibroblasts for 15 days.
Besides delivery regulation mechanisms there are in vitro transfection applications that treat cells as
microfactories to create pharmaceutical products. An example of using gene delivery to utilize cells as
manufacturing plants is the injection of engineered plasmids into silkworm eggs for the production of
type III procollagen [96]. This application is a useful way of producing protein-based polymers with
high sequence specificity and low polydispersity. This technology could be used to create scaffolds for
cell-seeding in tissue engineering applications.
14.6.2 Ex Vivo
Ex vivo gene therapy involves the methods of in vitro cell therapy coupled with the reintroduction of the
altered cells into an organism. Likely candidates for this approach are bone marrow progenitor cells of
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the adult (mesenchymal and hematopoetic) embryonic and adult stem cells. Delivery of the transfecting
(or transducing) gene can be accomplished via any of the methods mentioned previously in this chapter.
There has been much success with this application of gene therapy, which has progressed to the clinical
trial stage. Transplantation of nerve growth factor (NGF)-producing grafts of neural progenitor cells into
the septum and nucleus basalis of the rat cerebrum has been shown to diminish the atrophy of the brain
associated with aging [97]. Several more ex vivo investigations are outlined in Section 14.7 of this chapter.
14.6.3 In Vivo
To transport gene delivery complexes into living organisms, there exist several strategies that do not entail
surgery. The first method that might come to mind is injection via syringe. Injections can be very simple,
entailing introduction of complexes into a vein [98], into the peritoneum [99], or directly into a tissue such
as muscle [98] or tumor [100]. Even the blood–brain barrier can be circumvented via direct injection [101]
or injection into the jugular vein using the brain-specific promoter GFAP [102]. Additional approaches
for gene delivery to the central nervous system include injection into the cranial nerves for retrograde
transport of PEI/DNA complexes into the brainstem [103], and intraveneous administration of lipoplexes
conjugated with transferrin to effectively cross the blood–brain barrier via its transferrin receptors [104].
Permanent transfection via nonrandom integration has even been achieved via high-pressure tail vein
injection, in which plasmids coding for bacteriophage φC31 integrase were co-injected with plasmids
containing attB and a gene for human factor IX to yield site-specific genomic integration of the factor
IX gene in rat hepatocytes at native pseudo attP sites [105]. This concept could be applied to achieve
permanent transfection in many nonviral applications, which lend themselves to IV administration.
Another method of complex administration in vivo is through inhalation. Inhalation of aerosols is an
intuitive choice for gene delivery to the respiratory system. In mice, aerosol administration of a liposome–
DNA complex has yielded transfection results that persisted for 21 days [106]. More recently, aerosol
delivery of adeno-associated viral vectors through the nasal cavity produced positive transfection that was
apparent in the bloodstream in addition to that in the lungs [107]. Aerosol delivery is a logical method
of delivering transfection complexes to the lungs, while intravenous delivery might be better suited for
organs such as the spleen or liver, as demonstrated by results from a recent study that compared the two
delivery methods for PEI/DNA transfection complexes [108].
Mucosal administration of gene delivery complexes by oral or rectal routes is another option for in vivo
gene therapy. Mice that were fed chitosan-coated plasmid particles showed expression of the delivered
gene in the intestinal epithelium [109]. Another delivery method involves particle-mediated gene delivery
directly into the oral mucosa [110]. In the cited study, several marker and cancer-targeting genes were
delivered and shown to be expressed in the oral cavities of canines. Transrectal complex administration
(enema) has also been used to deliver genes to canines, where a recombinant adenovirus vector carrying
amarkergene(β-galactosidase) was used to demonstrate successful transduction in the colon [111].
14.7 Clinical Applications
To date, over 900 reported gene therapy clinical trials are underway worldwide; approximately 2% of these
are phase III investigations [112]. Well over half of the current trials utilize viruses as the gene delivery
vehicle. Physical and chemical (especially liposomal) delivery methods are also represented. Over half of
the current trials are cancer investigations [112]. Following is an overview of some of the more visible trials.
Severe combined immunodeficiency (SCID) was the first clinical application of gene therapy to go
into human trials, commencing in 1990. The treatment involved removing bone marrow stem cells,
altering them by exposure to healthy T-cells in culture, and then reintroducing the differentiated marrow-
derived cells to the host organism [113]. Another malady involving immunocompromise is the HIV
infection, a retrovirally caused disease that affects T-helper cells and can eventually lead to acquired
immunodeficiency syndrome (AIDS). Phase I, II, and III clinical trials are currently underway for several
applications of HIV-related gene therapy. Ex vivo manipulation of T lymphocytes with ribozymes, followed
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by reintroduction of the cells into the host, shows promise in that the T-cells have a greater survival time
and are clinically safe for the subject [114]. A separate study conducted on twins was completed in 2002,
and showed the safety and efficacy of ex vivo lymphocyte manipulation for expression of an anti-HIV gene
(revTD) [115].
Clinical investigations for tissue- or organ-related maladies are also underway. Ischemic heart disease
is the result of poor circulatory perfusion of cardiac muscle, commonly from coronary artery blockage.
The delivery of vascular endothelial growth factor (VEGF), first described as a tumor-produced vascu-
lar permeability factor, has been shown to stimulate angiogenesis in several tissues including cardiac
muscle [116]. Phase I studies have indicated that direct injection of naked [117] or adenovirally delivered
[118] plasmids encoding VEGF into cardiac tissue yields both safe and effective transfection/transduction.
Cystic fibrosis is another tissue/organ malady. The disease is the result of a mutation in a gene encoding
a cellular cAMP-mediated chloride channel, known as the cystic fibrosis transmembrane conductance
regulator (CFTR). With inadequate chloride transport capabilities, affected cells will develop an osmotic
gradient that is relieved by internalization of water from the extracellular environment. In cells that are
bathed in mucus, such as lung airway epithelia, the result is a loss of water from the coating mucus, raising
mucus viscosity and lowering the body’s ability to transport it via ciliary movement. Bacterial colonization
is often the result of such stasis. Clinical trials for the treatment of cystic fibrosis have been conducted
using different target administrations. A trial of liposome-mediated transfection for cystic fibrosis patients
showed the efficacy and safety of delivery of the CFTR gene to nasal epithelium cells [119]. Aerosolized
adenoviral particles carrying CFTR cDNA havealso been used clinically for transduction in the lungs [120].
Neurologic disorders have also been the target of clinical gene therapy trials. A phase I study of
Alzheimer’s disease involved the removal of primary fibroblasts from subjects, effectively transducing
the harvested cells with nerve growth factor and reintroducing the cells into the brain [121] (also reviewed
in Reference 122). The use of nerve growth factor to target the cholinergic neurons was employed with
the aim of preventing neural degradation and elevating levels of acetylcholine transferase.
Gene therapy applications for cancer treatment are very well represented in the group of clinical trial
investigations. Phase I and II trials directed at malignant gliomas are using a modified herpes virus to
target glioma cells in order to cause an immune response to diminish the number of cancerous cells [123].
The referenced study is currently focused on drug dose amount in subjects with recurrent glioblastoma.
A phase II study of ovarian cancer commenced in the year 2000 to determine the safety and efficacy
of genetically altered herpes simplex thymidine kinase-producing cells (HSV-TK) delivered into cancer-
affected ovaries [124]. The amount of cancer research that is underway is too large to allow a proper
presentation of the area in sufficient detail here.
14.8 Summary
Gene therapy is a very diverse and rapidly growing field. Researchers in many areas, including biology,
chemistry, physics, engineering, and medicine can find new applications for their creations and discoveries
in gene therapeutics. Since molecular biology and genetic recombination laid the foundation for controlled
genetic alteration, the field of gene therapy has expanded dramatically. The ability to alter cellular function
through the introduction of exogenous genetic material has drawn considerable hope that this technology
can be used to discover new disease treatments as well as explicate some of the mysteries of life at the
level of basic science. As laboratory and clinical trials proceed and knowledge of the area becomes more
rational and objective within the general public, gene therapy should prove to deliver beneficial and lasting
advances from the world of biomolecular science.
References
[1] Wolff, J. and Lederberg, J., An early history of gene transfer and therapy, Hum. Gene Ther., 5, 469,
1994.
mikos: “9026_c014” — 2007/4/9 — 15:51 — page 11 — #11
Gene Therapy 14-11
[2] Little E., et al., The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation,
and applications, Crit. Rev. Eukaryot. Gene Expr., 4, 1–18, 1994.
[3] Haider, M., Megeed, Z., and Ghandehari, H., Genetically engineered polymers: status and
prospects for controlled release, J. Control. Release, 95, 1, 2004.
[4] Sun, X., Wu, Z., and Hu, J., Suicide gene therapy of hepatocellular carcinoma and delivery
procedure and route of therapeutic gene in vivo, HPBD Int., 1, 373, 2003.
[5] Nakaya, T. et al., Decoy approach using RNA–DNA chimera oligonucleotides to inhibit the regulat-
ory function of human immunodeficiency virus type 1 Rev protein, Antimicrob. Agents Chemother.,
41, 319, 1997.
[6] Browning, C. et al., Potent inhibition of human immunodeficiency virus type 1 (HIV-1) gene
expression and virus production by an HIV-2 Tat activation-response RNA decoy, J. Virol., 73,
5191, 1999.
[7] Pace, B.S., Qian, X., and Ofori-Acquah, S.F., Selective inhibition of beta-globin RNA transcripts
by antisense RNA molecules, Cell. Mol. Biol., 50, 43, 2004.
[8] Li, M.J. et al., Specific killing of Ph+ chronic myeloid leukemia cells by a lentiviral vector-delivered
anti-bcr/abl small hairpin RNA, Oligonucleotides, 13, 401, 2003.
[9] Anderson, J. et al., Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4
siRNA, AIDS Res. Hum. Retroviruses, 19, 699, 2003.
[10] Elbashir, S.M., Lendeckel, W., and Tuschl, T., RNA interference is mediated by 21- and 22-
nucleotide RNAs, Genes Dev., 15, 188, 2001.
[11] Elbashir, S.M. et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured
mammalian cells, Nature, 411, 494, 2001.
[12] Miller, V.M. et al., Allele-specific silencing of dominant disease genes, Proc. Natl Acad. Sci. USA,
100, 7195, 2003.
[13] Miller, V. et al., Allele-specific silencing of dominant disease genes, Proc. Natl Acad. Sci. USA, 100,
7195, 2003.
[14] Douglas, J.T., Adenovirus-mediated gene delivery: an overview, Meth. Mol. Biol., 246, 3, 2004.
[15] Huser, A. and Hofmann, C., Baculovirus vectors: novel mammalian cell gene-delivery vehicles and
their applications, Am. J. Pharmacogenom., 3, 53, 2003.
[16] Boldogkoi, Z. and Nogradi, A., Gene and cancer therapy — pseudorabies virus: a novel research
and therapeutic tool? Curr.GeneTher., 3, 155, 2003.
[17] Goins, W.F. et al., Delivery using herpes simplex virus: an overview, Meth. Mol. Biol., 246, 257,
2004.
[18] Lu, Y., Recombinant adeno-associated virus as delivery vector for gene therapy—areview,Stem
Cells Dev., 13, 133 2004.
[19] Gafni, Y. et al., Gene therapy platform for bone regeneration using an exogenously regulated,
AAV-2-based gene expression system, Mol. Ther., 9, 587, 2004.
[20] Hodge, J.W. et al., Modified vaccinia virus ankara recombinants are as potent as vaccinia recom-
binants in diversified prime and boost vaccine regimens to elicit therapeutic antitumor responses,
Cancer Res., 63, 7942, 2003.
[21] Hu, Y. et al., Yaba-like disease virus: an alternative replicating poxvirus vector for cancer gene
therapy. J. Virol., 75, 10300, 2001.
[22] Cockrell, A.S. and Kafri, T., HIV-1 vectors: fulfillment of expectations, further advancements, and
still a way to go, Curr. HIV Res., 1, 419, 2003.
[23] Cheng, L. et al., Human immunodeficiency virus type 2 (HIV-2) vector-mediated in vivo gene
transfer into adult rabbit retina, Curr. Eye Res., 24, 196, 2002.
[24] Kafri, T., Gene delivery by lentivirus vectors an overview, Meth. Mol. Biol., 246, 367, 2004.
[25] Wolkowicz, R., Nolan, G.P., and Curran, M.A., Lentiviral vectors for the delivery of DNA into
mammalian cells, Meth. Mol. Biol., 246, 391, 2004.
[26] Rainov, N.G. and Ren, H., Clinical trials with retrovirus mediated gene therapy — what have we
learned? J. Neuro-oncol., 65, 227, 2003.
mikos: “9026_c014” — 2007/4/9 — 15:51 — page 12 — #12
14-12 Tissue Engineering
[27] Lilley, C. et al., Multiple immediate-early gene-deficient herpes simplex virus vectors allowing
efficient gene delivery to neurons in culture and widespread gene delivery to the central nervous
system in vivo, J. Virol., 75, 4343, 2001.
[28] Cao, B. and Huard, J., Gene transfer to skeletal muscle using herpes simplex virus-based vectors,
Meth. Mol. Biol., 246, 301, 2004.
[29] Billelo, J. et al., Transient disruption of intercellular junctions enables baculovirus entry into
nondividing hepatocytes, J. Virol., 75, 9857, 2001.
[30] Hu, Y. et al., Yaba-like disease virus: an alternative replicating poxvirus vector for cancer gene
therapy, J. Virol., 75, 10500, 2001.
[31] Garrett, D. et al., In utero recombinant adeno-associated virus gene transfer in mice, rats, and
primates, BMC Biotech., 3, 16, 2003.
[32] Ahmed, B. et al., Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction
of neurons using adeno-associated and lentiviral vectors, BMC Neurosci., 5, 4, 2004.
[33] Mastakov, M. et al., Immunological aspects of recombinant adeno-associated virus delivery to the
mammalian brain, J. Virol., 76, 8446, 2002.
[34] Zufferey, R., Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery, J. Virol.,
72, 9873, 1998.
[35] Curran, M. et al., Efficient transduction of nondividing cells by optimized feline immunodeficiency
virus vectors, Mol. Ther., 1, 31, 2000.
[36] Reiser, J. et al., Development of multigene and regulated lentivirus vectors, J. Virol., 74, 10589, 2000.
[37] Marschall, P., Malik, N., and Larin, Z., Transfer of YACs up to 2.3 Mb intact into human cells with
polyethylenimine, Gene Ther., 6, 1634, 1999.
[38] Vanderbyl, S., MacDonald, N., and de Jong, G., A flow cytometry technique for measuring
chromosome-mediated gene transfer, Cytometry, 44, 100, 2001.
[39] Rossenberg, S. van. et al., Stable polyplexes based on arginine-containing oligopeptides for in vivo
gene delivery, Nature, 11, 457, 2004.
[40] Chen, Q. et al., Branched co-polymers of histidine and lysine are efficient carriers of plasmids,
Nucleic Acids Res., 29, 1334, 2001.
[41] Toncheva V. et al., Novel vectors for gene delivery formed by self-assembly of DNA with poly(-
lysine) grafted with hydrophilic polymers, Biochim. Biophys. Acta, 1380, 354, 1998.
[42] Godbey, W., Wu, K., and Mikos A., Size matters: molecular weight affects the efficiency of
poly(ethylenimine) as a gene delivery vehicle, J. Biomed. Mater. Res., 45, 268, 1999.
[43] Fischer, D. et al., A novel non-viral vector for DNA delivery based on low molecular weight,
branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity,
Pharm. Res., 16, 1273, 1999.
[44] Tang, G. et al., Polyethylene glycol modified polyethylenimine for improved CNS gene transfer:
effects of PEGylation extent, Biomaterials, 24, 2351, 2004.
[45] Boussif, O. et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and
in vivo: polyethylenimine, Proc. Natl Acad. Sci. USA, 92, 7297, 1995.
[46] Manuel, W., Zheng, J., and Hornsby, P., Transfection by polyethyleneimine-coated microspheres,
J. Drug Target., 9, 15, 2001.
[47] Kuskowska-Latallo, J. et al., Efficient transfer of genetic material into mammalian cells using
Starburst polyamidoamine dendrimers, Proc. Natl Acad. Sci. USA, 93, 4897, 1996.
[48] Maruyama-Tabata, H. et al., Effective suicide gene therapy in vivo by EBV-based plasmid vector
coupled with polyamidoamine dendrimer, Gene Ther., 7, 53, 2000.
[49] Mao, H.Q. et al., Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and
transfection efficiency, J. Control. Release, 70, 399, 2001.
[50] Mansouri, S. et al., Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies
to improve transfection efficacy, Eur. J. Pharm. Biopharm., 57, 1, 2004.
[51] Ishii, T., Okahata, Y., and Sato, T., Mechanism of cell transfection with plasmid/chitosan
complexes, Biochim. Biophys. Acta, 1514, 51, 2001.
mikos: “9026_c014” — 2007/4/9 — 15:51 — page 13 — #13
Gene Therapy 14-13
[52] Padmanabhan, K. and Smith, T.A., Preliminary investigation of modified alginates as a matrix for
gene transfection in a HeLa cell model, Pharm. Dev. Technol., 7, 97, 2002.
[53] Gonzalez Ferreiro M. et al., Characterization of alginate/poly-l-lysine particles as antisense
oligonucleotide carriers, Int. J. Pharm., 239, 47 2002.
[54] Li, Z. et al., Controlled gene delivery system based on thermosensitive biodegradable hydrogel,
Pharm. Res., 20, 884, 2003.
[55] Girao Da Cruz, M., Simoes, S., and Pedroso De Lima, M., Improving lipoplex-mediated gene
transfer into C6 glioma cells and primary neurons, Exp. Neurol., 187, 65, 2004.
[56] Liu, F., Huang, L., and Liu, D., Factors controlling the efficiency of cationic lipid-mediated
transfection in vivo via intravenous administration, Gene Ther., 4, 517, 1997.
[57] Farhood, H., Serbina, N., and Huang, L., The role of dioleoyl phosphatidylethanolamine in
cationic liposome mediated gene transfer, Biochim. Biophys. Acta, 1235, 289, 1995.
[58] King, R., Gene delivery to mammalian cells by microinjection, Meth. Mol. Biol., 245, 167,
2004.
[59] Wilmut, I. et al., Viable offspring derived from fetal and adult mammalian cells, Nature, 385, 810,
1997.
[60] Hosaka, K. et al., Cloned mice derived from somatic cell nuclei, Hum. Cell, 13, 237, 2000.
[61] Neumann, E. et al., Gene transfer into mouse myeloma cells by electroporation in high electric
fields, EMBO J., 1, 841, 1982.
[62] Golzio, M., Teissié, J., and Rols, M., Direct visualization at the single-cell level of electrically
mediated gene delivery, Proc. Natl Acad. Sci. USA, 99, 1292, 2002.
[63] Mir, L. et al., High-efficiency gene transfer into skeletal muscle mediated by electric pulses, Proc.
Natl Acad. Sci USA, 96, 4262, 1999.
[64] Wang, Y. et al., Combination of electroporation and DNA/dendrimer complexes enhances gene
transfer into murine cardiac transplants, Am. J. Transplant., 1, 334, 2001.
[65] Schakowski, F. et al., Novel non-viral method for transfection of primary leukemia cells and cell
lines, Genet. Vaccines Ther., 2, 1, 2004.
[66] Sun, W. et al., In vivo cytokine gene transfer by gene gun reduces tumor growth in mice, Proc. Natl
Acad. Sci. USA, 92, 2889, 1995.
[67] Matsuno Y. et al., Nonviral gene gun mediated transfer into the beating heart, ASAIO J., 49, 641,
2003.
[68] Jeon, E., Kim, H.D., and Kim, J.S., Pluronic-grafted poly-(l)-lysine as a new synthetic gene carrier,
J. Biomed. Mater. Res., 66A, 854, 2003.
[69] Joubert, D. et al., A note on poly-l-lysine-mediated gene transfer in HeLa cells, Drug Deliv., 10,
209, 2003.
[70] Tan, P.H. et al., Transferrin receptor-mediated gene transfer to the corneal endothelium,
Transplantation, 71, 552, 2001.
[71] Van Rossenberg S.M. et al., Targeted lysosome disruptive elements for improvement of
parenchymal liver cell-specific gene delivery, J. Biol. Chem., 277, 45803, 2002.
[72] Godbey, W., Wu, K., and Mikos, A., Tracking the intracellular path of poly(ethylenimine)/DNA
complexes for gene delivery, Proc. Natl Acad. Sci. USA, 96, 5177, 1999.
[73] Godbey, W.T. et al., Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery,
J. Biomed. Mater. Res., 51, 321, 2000.
[74] Suh, J., Wirtz, D., and Hanes, J., Efficient active transport of gene nanocarriers to the cell nucleus,
Proc. Natl Acad. Sci. USA, 100, 3878, 2003.
[75] Brunner, S. et al., Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant
adenovirus, Gene Ther., 7, 7401, 2000.
[76] Escriou, V. et al., Critical assessment of the nuclear import of plasmid during cationic lipid-
mediated gene transfer, J. Gene Med., 3, 179, 2001.
[77] Zanta, M. et al., Gene delivery: a single nuclear localization signal peptide is sufficient to carry
DNA to the cell nucleus, Proc. Natl Acad. Sci. USA, 96, 91, 1999.
mikos: “9026_c014” — 2007/4/9 — 15:51 — page 14 — #14
14-14 Tissue Engineering
[78] Aronsohn, A.I. and Hughes, J.A., Nuclear localization signal peptides enhance cationic liposome-
mediated gene therapy, J. Drug Target., 5, 163, 1998.
[79] Bremner, K. et al., Factors influencing the ability of nuclear localization sequence peptides to
enhance nonviral gene delivery, Bioconjug. Chem., 15, 152, 2004.
[80] Parveen, Z. et al., Cell-type-specific gene delivery into neuronal cells in vitro and in vivo, Virology,
314, 74, 2003.
[81] White, S.J. et al., Identification of peptides that target the endothelial cell-specific LOX-1 receptor,
Hypertension, 37, 449, 2001.
[82] Argnani, R. et al., Specific targeted binding of herpes simplex virus type 1 to hepatocytes via the
human hepatitis B virus preS1 peptide, Gene Ther., 2004.
[83] Voinea, M., Binding and uptake of transferrin-bound liposomes targeted to transferrin receptors
of endothelial cells, Vascul. Pharmacol., 39, 13, 2002.
[84] Zhao, X.B. and Lee, R.J., Tumor-selective targeted delivery of genes and antisense oligodeoxyribo-
nucleotides via the folate receptor, Adv. Drug Deliv. Rev., 56, 1193, 2004.
[85] Yang, Q. et al., Development of novel cell surface CD34-targeted recombinant adenoassociated
virus vectors for gene therapy, Hum. Gene Ther., 9, 1929, 1998.
[86] Kunath, K. et al., Galactose–PEI–DNA complexes for targeted gene delivery: degree of substitution
affects complex size and transfection efficiency, J. Control. Release, 88, 159, 2003.
[87] Wilcox, D.A. et al., Integrin alphaIIb promoter-targeted expression of gene products in mega-
karyocytes derived from retrovirus-transduced human hematopoietic cells, Proc. Natl Acad. Sci.
USA, 96, 9654, 1999.
[88] Park, C. et al., Targeting of therapeutic gene expression to the liver by using liver-type pyruvate
kinase proximal promoter and the SV40 viral enhancer active in multiple cell types, Biochem.
Biophys. Res. Commun., 314, 131, 2004.
[89] Godbey, W.T. and Atala, A., Directed apoptosis in Cox-2-overexpressing cancer cells through
expression-targeted gene delivery, Gene Ther., 10, 1519, 2003.
[90] Ikegami, S. et al., Treatment efficiency of a suicide gene therapy using prostate-specific membrane
antigen promoter/enhancer in a castrated mouse model of prostate cancer, Cancer Sci., 95, 367,
2004.
[91] Itakura, K. et al., Expression in Escherichia coli of a chemically synthesized gene for the hormone
somatostatin, Science, 198, 1056, 1977.
[92] Lee, J. et al., In vitro model for liposome-mediated adenomatous polyposis coli gene transfer in a
duodenal model, Dis. Col. Rec., 47, 219, 2004.
[93] Krosl, J., In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein, Nat.
Med., 9, 1428, 2003.
[94] Li, R.H. et al., Dose control with cell lines used for encapsulated cell therapy, Tissue Eng., 5, 453,
1999.
[95] Huang, Y.C. et al., Fabrication and in vitro testing of polymeric delivery system for condensed
DNA, J. Biomed. Mat. Res., 67A, 1384, 2003.
[96] Tomita, M. et al., Transgenic silkworms produce recombinant human type III procollagen in
cocoons, Nat. Biotechnol., 21, 52, 2003.
[97] Martínez-Serrano, A. et al., Ex vivo nerve growth factor gene transfer to the basal forebrain in
presymptomatic middle-aged rats prevents the development of cholinergic neuron atrophy and
cognitive impairment during aging, Proc. Natl Acad. Sci. USA, 95, 1858, 1998.
[98] Magin-Lachmann, C. et al., In vitro and in vivo delivery of intact BAC DNA — comparison of
different methods, J. Gene Med., 6, 195, 2004.
[99] Brown, C.B., Use of the peritoneal cavity for therapeutic delivery, Perit. Dial. Int., 19, 512, 1999.
[100] Shariat, S.F., Adenovirus-mediated transfer of inducible caspases: a novel “death switch” gene
therapeutic approach to prostate cancer, Cancer Res., 61, 2562, 2001.
[101] Xia, C.F. et al., Kallikrein gene transfer protects against ischemic stroke by promoting glial cell
migration and inhibiting apoptosis, Hypertension, 43, 452, 2004.
mikos: “9026_c014” — 2007/4/9 — 15:51 — page 15 — #15
Gene Therapy 14-15
[102] Shi, N. et al., Brain-specific expression of an exogenous gene after i.v. administration, Proc. Natl
Acad. Sci. USA, 98, 12754, 2001.
[103] Wang, S. et al., Transgene expression in the brain stem effected by intramuscular injection of
polyethylenimine/DNA complexes, Mol. Ther., 3, 658, 2001.
[104] Shi, N. and Pardridge, W., Noninvasive gene targeting to the brain, Proc. Natl Acad. Sci. USA, 97,
7567, 2000.
[105] Olivares, E.C. et al., Site-specific genomic integration produces therapeutic factor IX levels in mice,
Nat. Biotechnol., 20, 1124, 2002.
[106] Stribling, R. et al., Aerosol gene delivery in vivo, Proc. Natl Acad. Sci. USA, 89, 11277, 1992.
[107] Auricchio, A. et al., Noninvasive gene transfer to the lung for systemic delivery of therapeutic
proteins, J. Clin. Invest., 110, 499, 2002.
[108] Koshkina, N.V. et al., Biodistribution and pharmacokinetics of aerosol and intravenously admin-
istered DNA-polyethyleneimine complexes: optimization of pulmonary delivery and retention,
Mol. Ther., 8, 249, 2003.
[109] Chen, J. et al., Transfection of mEpo gene to intestinal epothelium in vivo mediated by oral delivery
of chitosan-DNA nanoparticles, World J. Gastroenterol., 10, 112, 2004.
[110] Keller, E.T. et al., In vivo particle-mediated cytokine gene transfer into canine oral mucosa and
epidermis, Cancer Gene Ther., 3, 186, 1996.
[111] Weld, K.J. et al., Transrectal gene therapy of the prostate in the canine model, Cancer Gene Ther.,
9, 189, 2002.
[112] Journal of Gene Medicine Website, http://www.wiley.co.uk/genmed/clinical, Accessed 10/18/2005.
[113] Cavazzana-Calvo, M. et al., Gene therapy of human severe combined immunodeficiency (SCID)-
X1 disease, Science, 288, 627, 2000.
[114] Wong-Staal, F., Poeschla, E.M., and Looney, D.J., A controlled, phase 1 clinical trial to evaluate
the safety and effects in HIV-1 infected humans of autologous lymphocytes transduced with a
ribozyme that cleaves HIV-1 RNA, Hum. Gene Ther., 9, 2407, 1998.
[115] Twins Study of Gene Therapy for HIV Infection, National Human Genome Research Institute
(NHGRI).
[116] Senger, D.R. et al., Tumor cells secrete a vascular permeability factor that promotes accumulation
of ascites fluid, Science, 219, 983, 1983.
[117] Losordo, D. et al., Gene therapy for myocardial angiogenesis: initial clinical results with direct
myocardial injection of phVEGF165 as sole therapy for myocardial ischemia, Circulation, 98,
2800, 1998
[118] Rosengart, T.K. et al., Six-month assessment of a phase I trial of angiogenic gene therapy for the
treatment of coronary artery disease using direct intramyocardial administration of an adenovirus
vector expressing the VEGF121 cDNA, Ann. Surg., 230, 466, 1999.
[119] Sorscher, E., Study chair, Phase I Study of Liposome-Mediated Gene Transfer in Patients with
Cystic Fibrosis, University of Alabama.
[120] Moss, R.B. et al., Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis
transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter,
double-blind, placebo-controlled trial, Chest, 125, 509, 2004.
[121] Tuszynski, M., Prin. invest., Gene therapy for Alzheimer’s Disease Clinical Trial, University of
California, San Diego.
[122] Tuszynski, M.H. and Blesch, A., Nerve growth factor: from animal models of cholinergic neuronal
degeneration to gene therapy in Alzheimer’s disease, Prog. Brain Res., 146, 441, 2004.
[123] de Haan, Hans, Prin. invest., Gene Therapy in Treating Patients with Recurrent Malignant Glioma,
University of Alabama at Birmingham Comprehensive Cancer Center, Birmingham, AL.
[124] Link, C., Prin. invest., Gene Therapy in Treating Women With Refractory or Relapsed Ovarian
Epithelial Cancer, Fallopian Tube Cancer, or Peritoneal Cancer, Human Gene Therapy Research
Institute, Des Moines, Iowa.